Figure 3.

Hyaluronidase (HYAL)-2 from cell lysates (CL) of myofibroblasts has no enzymatic action in hyaluronan (HA)-degradation assays. Fibroblasts were grown to 70% confluence and were transiently transfected with pCR3.1 containing HYAL1 or HYAL2 coding regions. Control transient transfections were performed with the corresponding empty vector. Cells were growth-arrested before incubation in serum-free medium containing 10 ng/mL transforming growth factor beta 1 (TGFB1; TGF-β1). A: Total cellular RNA was extracted, purified, and analyzed by RT-PCR, and the products were separated on 3% agarose gels. Controls were negative (−ve) RT and negative (−ve) PCR, where no polymerase enzyme was added to the reaction mixture. B: HA substrate gel zymography of HYAL activity. The medium was collected, and the cells were extracted in lysis buffer. The HYAL activity was isolated from the conditioned medium (CM) and CL by DEAE-Sephacel ion exchange and lyophilized. Positive controls consisted of diluted human serum. Polyacrylamide gel electrophoresis was performed without SDS in a 7.5% polyacrylamide gel (+HA) and visualized by Alcian Blue and Coomassie Brilliant Blue stain. C: HYAL activity (circles) of CM and CL was further analyzed by incubating with high–molecular-weight [³H]-HA. HA size distribution was examined and compared with the original [³H]-HA preparation, incubated in the absence of the conditioned medium or CL concentrate (squares). All images are representative of four individual experiments. GADPH, glyceraldehyde phosphate dehydrogenase.